The goal of our work is the structural elucidation of the large multiprotein assemblies involved in RNA polymerase II transcription. We have isolated and performed preliminary structural studies on a chromatin- remodeling complex (RSC), general transcription factors, and Mediator. Specific aims for the project period are structure determination of (1) general transcription factor H, (2) RNA polymerase II - general transcription factor complexes, (3) Mediator and Mediator - RNA polymerase II complexes, and (4) RSC and RSC - nucleosome complexes. Our efforts will culminate in the structure determination of a complete 48-protein RNA polymerase II transcription initiation complex, and of a RSC-nucleosome complex in the transcriptionally activated state. In view of the size of these complexes, almost all greater than one million Daltons, structures will be determined by cryoelectron microscopy and image processing. Wherever possible, 2-D crystals will be formed on lipid layers, permitting image processing by Fourier analysis. In the absence of crystals, image processing will be done by single particle analysis. The results should reveal the molecular basis of transcriptional activation, initiation,and regulation. They will be directly relevant to human disease. For example, mutations in subunits of factor H are responsible for a number of inherited human disease syndromes. Mutations in RSC or RSC-related proteins are implicated in oncogenesis. =>ERFORMANCE SITE(S) (organization, city, state) Stanford University School of Medicine Stanford, California 94305-5126 KEY PERSONNEL. See instructions on Page 11. Usecontinuationpagesas neededto provide the required information in the format shown below. Name Organization Role on Project ROGER D. KORNBERG STANFORD UNIVERSITY P.I. DAVID A. BUSHNELL STANFORD UNIVERSITY POSTDOC FELLOW PHS 398 (Rev. 4/98) Page2 BB Number pages consecutively at the bottom throughout the application. Do ngiuse suffixes such as 3a, 3b. CC ^ropal Investigator/Program Director (Last, first, middle^* Kornberg. Roger D. Type the name of the principal investigator/program director at the top of each printed page and each continuation page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page Description,